Epithelial cell attachment and adhesion protein expression on novel in sol TiO2 coated zirconia and titanium alloy surfaces

Abstract An adequate mucosal attachment is important when it comes to preventing peri‐implant inflammation. The aim of this study was to compare epithelial cell adhesion and adhesion protein expression on in sol TiO2‐coated and non‐coated zirconia and titanium alloy surfaces. Fifty‐six zirconia and titanium discs were cut, and half of them were coated with bioactive TiO2‐coating. To study the epithelial cell attachment, human gingival keratinocytes were cultivated on discs for 1, 3, 6, and 24 h. The cell proliferation was detected by cultivating cells for 1, 3, and 7 days. In addition, the levels of adhesion proteins laminin y2, integrin α6, β4, vinculin, and paxillin were detected with Western Blot method. Furthermore, high‐resolution imaging of the actin cytoskeleton and focal adhesion proteins was established. Longer‐term cell culture (1–7 days) revealed higher cell numbers on the coated zirconia and titanium discs compared to non‐coated discs. The difference was statistically significant (p < .05) after 24 h on coated zirconia and after 3 and 7 days on coated titanium discs compared to non‐coated discs. Clear induction in the protein levels of laminin y2 and integrin α6 were detected on both coated samples, meanwhile integrin β4 were clearly induced on coated titanium alloy. The microscope evaluation showed significantly increased cell spreading on the coated discs. According to this study, the in sol induced TiO2‐coating increases keratinocyte attachment and the expression of adhesion proteins on coated zirconia and titanium in vitro. Consequently, the coating has potential to enhance the mucosal attachment on implant surfaces.

of a natural tooth. 6 Peri-implant mucosa consists of connective tissue covered by epithelium. The epithelium attaches to the tooth and the implant surface via hemidesmosomes and extra cellular matrix (ECM) called the internal basal lamina. 7,8 Laminin-332, composed of α3, β3, and γ2 chains, is an important adhesion protein localized in the basal lamina. 9 Another important part in hemidesmosomes is integrin α6β4, which penetrate through cell membrane and binds to basal lamina protein laminin-332. Integrin α6β4 has been proved to be an important factor in epithelial attachment. 10,11 Previous studies have indicated that the connective tissue under epithelium is not properly attached to the implant or abutment surface but rather the gingival fibers circle the implant abutment parallery, not attaching it directly but forming a connective tissue capsule. 12 As the epithelial attachment forms the first barrier against bacteria, it plays a crucial role in preventing peri-implant inflammation. In this study, we focus on to study the epithelial attachment via hemidesmosomes to implant surface.
Means to ensure a proper soft tissue attachment have been studied previously. One option to enhance the mucosal attachment and adhesion protein adsorption is through surface modifications, for example, by using different sol-gel derived coatings. 13 The benefits of sol-gel derived TiO 2 -dip coatings are, that it is nonresorbable, thin and easy to produce. 13 However, the utilization of sol-gel processes is limited, because the flexibility to operate is restricted to only a few kinds of shapes. In fact, more complicated 3D structures require alternative surface treatment methods. In this study, we used modified nanoporous bioactive TiO 2 -coating that can change surface hydrophilicity, make it biologically active and enhance cell adhesion to the implant surface. In the present study, a novel in sol polycondensation induced TiO 2 -coating, with a simple coating procedure was evaluated.
Instead of dip coating the coatings were produced by direct gelation onto implant surfaces within the sol.
Both zirconia and titanium alloy are frequently used in oral implant abutments. Titanium has been the most used implant material due to its favorable properties, such as biocompatibility and good mechanical strength. 14 As demands for aesthetic treatment results have increased, zirconia has become an increasingly popular abutment material especially in aesthetic regions. Zirconia fulfills many properties that are expected from implant materials. The good biocompatibility, flexural strength and color that is very similar to natural teeth are ones to name. 15,16 Above all, the most important thing for good aesthetic result is to prevent the gingival recession from happening, which can be accomplished by ensuring sufficient soft tissue volume and a proper formation of mucosal attachment. 17 The aim of this study was to compare the effect of TiO 2 -coating on cell attachment and expression of adhesion proteins on coated and non-coated titanium and zirconia surfaces. The study was carried out by measuring the amounts of adhered cells, analyzing the cell spreading and the deposition of focal adhesion proteins using confocal microscopy and Western Blot-method. The hypothesis was, that the TiO 2 -coating would enhance cell adhesion, proliferation and the expression of adhesion molecules.
Thereafter, both zirconia and titanium discs were washed sonically first with acetone, then with ethanol, for 5 min.

| Cell cultures
All the samples were sterilized in an autoclave before the cell cultures.
In this research, spontaneously immortalized human gingival keratinocytes (GK) were used. The GK were earlier collected from a human gingival biopsy sample. 20 The cells were blended in keratinocyte-serum-free medium (SFM) (Gibco ® , Thermo Fisher, USA) and cultured on the samples at a density of 25,000 cells/cm 2 .

| Cell adhesion
GK were cultured on zirconia and titanium discs for 1, 3, 6, and 24 h to define the cell adhesion on each surface. The cells were incubated at 37 C in full media and after a certain time period, washed with phosphate-buffered saline (PBS). The discs were handled with TE-buffer (10 mmol/L Tris, 1 mmol/L EDTA) and the solution was stored at À70 C. Next, the solution was melted and sonicated for 30 seconds each. Thereafter, the samples were treated with fluorescent nucleic acid stain (Pico-Green dsDNA, Molecular Probes Europe).
One-hundred microliters of solution from each sample was used to define the fluorescence values using wavelength of 490 and 535 nm (BioTek synergy HT). The total amount of DNA was calculated by comparing the fluorescence values to the standard curve.

| Cell proliferation
Cell proliferation was studied by growing GK on the discs for 1, 3, and 7 days after which the specimens were handled with Alamar Bluereagent (Thermo Fischer, USA) mixed in SFM. Thereafter, the discs were incubated for 3 h in a CO 2 -incubator at 37 C degrees. Subsequently, 200 μl of the solution from every specimen was pipetted on a microtiter plate and cell amounts were defined by measuring the absorbance of the samples with the wavelengths of 569 nm and 594 nm (Multiskan FC, Thermo Scientific) and the given values were compared to the standard curve. The relative cell attachment was defined by comparing cell amounts of the coated samples to the values of the control groups.
The samples from Day 1 were fixed with 4% paraformaldehyde for 15 min, after which the samples were washed with PBS and stored at +4 C prior to staining for microscopy.

| Data analyses
The data was analyzed and the graphs were made with GraphPad Prism-program. The unpaired T-test and Mann-Whitney U-test was used in calculating the p-values. Confocal microscope images

| SEM analyses
The surface topography of coated and non-coated discs was evaluated using 50,000x magnification. SEM images revealed small, nanostructure particles on coated surfaces ( Figure 1A). The particles are similar in size and shape as on titanium surfaces proven to improve cell and tissue adhesion in earlier studies. 21 EDS-analyze confirmed the presences of titanium particles on coated zirconia samples, meanwhile non-coated samples were titanium-free ( Figure 1B).

| TiO 2 coating enhances cell attachment
To determine initial cell adhesion efficiency, short-time plating assays were performed. The amount of cells attached, determined by DNA amounts, was significantly greater with TiO 2 -coated zirconia compared to non-coated zirconia (p < .05) after 24 h ( Figure 2B). When comparing titanium and zirconia samples, the adhesion was significantly increased on zirconia samples after 1, 3, and 6 h ( Figure 2C).
In order to study if cell proliferation would be higher on coated surfaces, the cell numbers were measured after 1, 3, and 7 days and relative cell attachment was calculated. A significant difference (p < .05) was found between coated and non-coated zirconia samples after the first day and between coated and non-coated titanium samples after 3 and 7 days (Figure 3). No significant difference was detected between zirconia and titanium samples.

| TiO 2 coating induces laminin and integrin protein expression
To determine whether the increased cell attachment could be explained by the induction of adhesion protein expression total protein levels were determined. In Western Blot analyses, there was significant induction of laminin y2 and integrin α6 on the coated zirconia and titanium samples. The level of integrin β4 was also significantly higher on coated titanium samples compared to non-coated titanium. Meanwhile a clear difference was not found between coated and non-coated zirconia samples. Paxillin and vinculin levels were significantly higher on the coated zirconia surface compared to the non-coated samples. Concerning the titanium samples, paxillin and vinculin levels were more equal between coated and non-coated surfaces (Figure 4).

| Confocal microscope analyses
In order to study if increased adhesion protein expression correlated with increased cell spreading, confocal microscope analysis of actin was performed. Cell spreading was significantly higher on both coated zirconia and titanium discs compared to non-coated controls indicating a faster cell attachment on both coated materials ( Figure 5).
The adhesion proteins laminin γ2, integrin α6 and β4 were located peripheral at hemidesmosomes indicating that these proteins would participate in epithelial cell adhesion. In turn, the expression of vinculin and paxillin was mostly seen diffusely in the cytoplasm. Figure 6 demonstrates the expression of adhesion proteins on the bottom layer of the cells (Figure 6A-L). The orthogonal view of the samples confirms, that laminin γ2, integrin α6 and β4 had a higher signal on the bottom layer of the cell. Whereas vinculin and paxillin were located more diffusely around the cell ( Figure 7B). When measuring the signal volume of laminin γ2, integrin α6 and β4 on the bottom layer of the cells, the volume was higher on the coated samples. The difference was significant concerning integrin α6 on titanium samples and integrin β4 and laminin γ2 on zirconia samples ( Figure 7A).

| DISCUSSION
This study revealed that novel in sol induced TiO 2 -coating enhances epithelial cell adhesion, adhesion molecule expression and cell spreading on both zirconia and titanium surfaces in vitro. Especially laminin y2, integrin α6 and β4 subunits were clearly induced in keratinocytes cultured on coated samples. This is notable, because these Lm-332 and integrin α6β4 are important elements in epithelial attachment via hemidesmosomes. 10 The study clearly shows that bioactive TiO 2 -coating can be effective at the molecular level.
The results are promising and support the set hypothesis. The study is in line with earlier studies concerning sol-gel derived oven sintered TiO 2 -coating. Previously, Meretoja et al. 22 23 However, use of sol-gel derived TiO 2 coatings has been limited due to difficulties in industrial level production of the coatings.
In cultured keratinocytes on both zirconia and titanium samples, the expression of paxillin and vinculin was mostly cytoplasmic, so called non-focal diffuse punctate distribution. Earlier studies have indicated that paxillin and vinculin are important proteins in focal adhesions. 24,25 Vinculin has many roles in cell signaling, and it is actin-binding protein that faces the integrin mediated focal adhesion and this way can affect to the cell attachment. 26 A result of this study could indicate, that oral keratinocytes attach to the implant surface mainly via hemidesmosomes rather than focal adhesions, as focal adhesion proteins were rarely seen in focal adhesion spots. In Western Blotting, there was a significant difference in vinculin and paxillin levels between coated and non-coated zirconia samples.
However, the microscope images revealed that these proteins would not be in central role in epithelial cell adhesion. Instead, this study shows that laminin γ2 and integrin α6β4 are located in hemidesmosomes, which indicates that these proteins have an important role in cell adhesion to substratum as earlier studies have also stated. 10,27 This study demonstrates that the in sol induced bioactive TiO 2coating enhances the levels of adhesion proteins and increases cell spreading. The cell spreading is improved supposedly due to the more hydrophilic surface of coated materials. The amount of epithelial cells was higher on the coated samples at the first time points. After 2 days, the cell amounts became quite even. This could mean that coating improves initial cell attachment, but does not influence cell proliferation.
In earlier studies, a sol-gel dip-coating method has been used. 13,22,23,28 Dip coating has its challenges when coating more complicated shaped objects. This new "coating made in sol"-technique makes it possible to coat more complex 3D objects. It takes less time and more pieces can be coated in the same time making coating process more efficient. Another potential way to coat implant abutments, is the hydrothermal technique. 29,30 Earlier studies have also shown that UV treatment is able to make the coating even more hydrophilic and could improve the cell attachment even more. 30,31 Moreover, the TiO 2 -coating does not only enhance the surface hydrophilicity but also changes surface topography by making it smooth and nanoporous. The effects of surface treatments on cell attachment have been widely studied and smooth surface has been found to be more favorable for cell attachment. 32 The TiO 2 -coating is able to induce calcium phosphate growth on its surface, which has been thought to partly explain favorable soft tissue reactions to TiO 2coated surface. 13 Although the TiO 2 -coating is beneficial for cell adhesion, studies have indicated that it does not have the same kind effect to oral bacteria, so the coating does not promote bacterial colonization. 33 According to this study, in sol induced TiO 2 -coating functioned in a similar manner on titanium alloy and zirconia surfaces. On both materials, the coating had a positive effect on the cell attachment and protein expression. During the first hours, the adhesion seemed to be faster on the zirconia surface. Nevertheless, the difference between titanium and zirconia became quite even in later time points. Some previous studies have indicated titanium being a better choice when it comes to mucosal attachment. Atsuta et al. 34 demonstrated epithelial sealing to titanium and zirconia surfaces. In their study, the epithelial bond was weaker and there were only few adhesive structures on the zirconia surface when compared to titanium samples. In the same study, the levels of adhesion proteins integrin β4 and plectin were lower on zirconia samples. These results are contradictory, as our study indicated a similar response between zirconia and titanium samples. On the other hand, there are also studies that have suggested zirconia to be the material of choice when a decent soft tissue attachment is wanted. Lee et al. 35 studied healing patterns between zirconia and titanium. The results indicated a higher expression of adhesion molecules and a more desirable biological width around zirconia abutments. All in all, both zirconia and titanium have their pros and cons.
According to this study, there seems no significant difference between these materials when it comes to epithelial cell attachment, but nanoporous TiO 2 -coating seems to enhance the epithelial cell function on both material surfaces.

| CONCLUSION
To conclude, the novel in sol induced TiO 2 -coating seems to have favorable effects on epithelial cell adhesion and cell spreading when compared to zirconia and titanium without surface treatment. This could potentially improve the formation of soft tissue attachment to implant or abutment surface. However, in vivo studies are needed to prove the real potential of the coating.

DATA AVAILABILITY STATEMENT
Original data are available on request from the corresponding author.